Cell culture and reagents
The human embryonic kidney HEK293T cells, normal mammary epithelial Hs578Bst cells and breast cancer Hs578T, MDA-MB-231, MDA-MB-468, MCF7, and BT549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Breast cancer SW527 cells were provided by Shanghai Sciencelight Biology Science & Technology Co. Ltd (Shanghai, China). HEK293T, Hs578Bst, Hs578T, MCF7, and SW527 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin in 5% CO2 at 37 °C. BT549 cells were cultured in RPMI-1640 medium with 10% FBS. MDA-MB-231 and MDA-MB-468 cells were grown in Leibovitz’s L-15 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a humidified incubator without CO2. All antibiotics were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA). All chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) except for special instructions.
Plasmids
The expression plasmids for C-terminally Flag-tagged and His-tagged TMEM97, PGRMC1 and LRP5 were purchased from WZ Bioscience Inc. The SuperTOPFlash reporter plasmid was provided by Karl Willert, University of California, San Diego. The AP1-Luc and NFAT-Luc reporter vectors, the expression plasmids encoding Wnt1, Wnt3, Fzd5, Fzd7, LRP6, β-catenin, DVL2, and pCMXβgal (β galactosidase, β-gal) have been described previously [10]. The 8×GTIIC-Luc reporter plasmid was purchased from Addgene (Cambridge, MA, USA). For the construction of C-terminally V5-tagged LRP6, CK1δ and CK1ε expression plasmids, the cDNA encoding LRP6, CK1δ, and CK1ε was amplified by RT-PCR and cloned into the pcDNA 3.1/V5-His mammalian expression vector. The N-terminally tagged Flag-CK1ε and Flag-CK1δ expression plasmids were constructed by inserting the corresponding cDNA into the pFlag-CMV2 expression vector. The dominant-negative CK1α, CK1γ, CK1δ, and CK1ε, in which a lysine at position 38 was mutated to arginine [20], were generated by site-directed mutagenesis according to the manufacturer’s instructions (Easy Mutagenesis System, TransGen Biotech, Beijing, China). For the construction of His-tagged TMEM97, GST-tagged CK1ε, and GST-tagged LRP6 intracellular fragment (amino acids 1394-1613), the cDNAs encoding human TMEM97, CK1ε, and LRP6 intracellular domain were amplified by PCR. The resulting PCR product of TMEM97 was then subcloned into pET-28 a (+) vector. CK1ε and LRP6 intracellular domain were subcloned into the expression vector pGEX3X. The resulting plasmids were designated pHis-TMEM97, pGST-CK1ε, and pGST-LRP6/1394-1613, respectively. LRP6 mutant lacking the extracellular domain (LRP6-ΔN) was constructed with pFlag-CMV2 expression vector by deleting 1245 amino acids in the N-terminus of LRP6 according to the method described previously [21]. The pEGFP-N1 or pmCherry-N1 vectors were used in constructing the LRP6-GFP or TMEM97-mCherry expression plasmids, respectively. All constructs were verified by DNA sequencing.
Generation of TMEM97 knockout cell lines by CRISPR-Cas9
TMEM97 was knocked out in Hs578T and MDA-MB-231 cell lines by using CRISPR/Cas9 technology as previously described [22]. Briefly, the single-guide RNA (Table S1) was cloned into the LentiCRISPRv2 vector (Addgene, Cambridge, MA, USA) to obtain the TMEM97 CRISPR/Cas9 KO plasmid. Using the LentiCRISPRv2 vector as a control, Lentivirus was produced by using 10 µg TMEM97 CRISPR/Cas9 KO plasmid or control CRISPR/Cas9 plasmid and two packaging plasmids including 2.5 μg pMD2.G (Addgene, Cambridge, MA, USA) and 7.5 µg psPAX2 (Addgene, Cambridge, MA, USA) in HEK293T cells with 60 µL Lipofectamine 2000 transfection reagent under standard conditions. Media was changed after 12 h of transfection, and the cells were cultured for 2 days. The supernatant was collected and centrifugated at 20,000 rpm for 2 h at 4 °C to harvest virus particles. Virus was immediately added to Hs578T and MDA-MB-231 cells with 8 µg/mL polybrene. After 72 h of infection, cells were then selected for stable expression of cas9 in the presence of 3 µg/mL puromycin (Thermo Fisher Scientific, Waltham, MA, USA) for one week. The puromycin-resistant stable clones were pooled and TMEM97 deficiency was confirmed by Western blotting analysis. Pooled clones and their parental clones were used to examine their biological behaviors.
In order to verify stable TMEM97 gene knockout in MDA-MB-231 cells, the target sites were amplified from the extracted DNA of CRISPR-transduced MDA-MB-231 cells, and amplicons were sequenced. The genomic DNA sequences spanning the target site in parental and TMEM97 knockout MDA-MB-231 cells were presented in Fig. S1 (Fig. S1).
Luciferase reporter gene assays
HEK293T cells were transfected in 24-well plates with the SuperTOPFlash reporter plasmid, control plasmid for β-gal and the indicated amounts of expression plasmids using Lipofectamine 2000 according to the manufacturer’s instructions. Luciferase assays were conducted using a luciferase assay kit (Promega, Shanghai, China). The luciferase values were normalized using a β-gal internal control to determine the variation in transfection efficiency.
Mass spectrometry assays
The mass spectrometry assay was performed as described previously [23]. Briefly, HEK293T cells were transfected with N-terminally Flag-tagged LRP6-ΔN expression vector or empty vector. The total protein of the cells was harvested with RIPA buffer containing 50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, protease inhibitors (Bimake, Beijing, China), and phosphatase inhibitors (Topscience, Shanghai, China), followed by centrifugation. Immunoprecipitations were performed using anti-Flag agarose beads (Bimake, Beijing, China) incubated with the supernatant overnight at 4 °C. The beads were washed three times with RIPA buffer. Beads with extracted proteins were digested by trypsin (Promega, Shanghai, China). The peptide sequences from these proteins were then extracted for mass spectrometry analysis on a Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA).
Quantitative real-time PCR analyses
Total RNA was extracted by RNAiso Plus (TaKaRa, Beijing, China) and subsequently reverse-transcribed into cDNA using the Primescript RT Reagent Kit (TaKaRa, Beijing, China) according to the manufacturer’s instructions. Quantitative PCR analysis was carried out with 2× SYBR Green qPCR Master Mix (Promega, Shanghai, China). The primer sequences are listed in Table S1.
Immunoblot analyses
Cells or tumor tissues were lysed in RIPA buffer, followed by sonication. The lysates were quantified by a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to poly-vinylidenefluoride (PVDF) membranes. Western blotting was performed with the indicated primary antibodies at 4 °C overnight. Then the PVDF membranes were incubated with secondary antibodies for 1 h at room temperature. After incubation with ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, Shanghai, China), the immunoblots were developed by the Tanon 5200 Chemiluminescent Imaging System (Tanon Science and Technology, Shanghai, China). The primary and secondary antibodies are given in Table S2.
Co-immunoprecipitation assays
Total cell lysates were harvested in 500 µL RIPA buffer with freshly added protease inhibitor and phosphatase inhibitor cocktails, followed by centrifugation at 12,000 rpm for 15 min at 4 °C. The lysates were quantified and incubated with respective antibodies and protein G beads at 4 °C overnight. Immunoprecipitations were conducted using anti-Flag or anti-V5 agarose beads (Bimake, Beijing, China), respectively. The beads were washed four times with RIPA buffer. Proteins were eluted by boiling the samples in SDS loading buffer and analyzed by SDS-PAGE and immunoblotting.
GST pull-down assays
His-TMEM97, GST, and GST-LRP6/1394-1613 fusion proteins were expressed in Escherichia coli (E. coli) BL21 and the expression of fusion proteins was induced with IPTG (Sigma-Aldrich, Shanghai, China) at 16 or 28 °C for 8–12 h. His-TMEM97 was purified by NiNTA Purification System while GST and GST-fusion proteins were purified by affinity chromatography with glutathione-sepharose 4B beads (GE Healthcare, Piscataway, NJ, USA). For the in vitro pull-down assay, His-TMEM97 fusion protein was incubated with GST or GST-LRP6/1394-1613 fusion proteins at 4 °C rotating for 4 h, then pulled down with glutathione-sepharose 4B beads. The immunoprecipitates were washed five times with PBS buffer. The beads were boiled in 95 °C with SDS loading buffer and analyzed by SDS-PAGE and immunoblotting.
Confocal imaging assays
HEK293T cells cultured on coverslips were transfected with TMEM97-mCherry expression plasmid alone or in combination with LRP6-GFP expression vector. After 48 h of transfection, cells were fixed with 4% paraformaldehyde at room temperature for 15 min, the cell nucleus was stained with DAPI (4,6-diamidino-2-phenylindole) and cells were mounted. The slides were observed with a fluorescence microscope (LSM880, ZEISS, Germany) at the Instrumental Analysis Center of Shenzhen University.
Cell viability assays
TMEM97 knockout Hs578T and MDA-MB-231 cells and their parental wild-type counterparts were seeded at 1×103 cells/well in 96-well plates. MTT reagent (Sangon Biotech, Shanghai, China) (5 mg/mL, 20 µL/well) was added after 24 h, 48 h, and 72 h of culture, and then incubated for another 4 h. The formazan crystals were dissolved in 100 µL DMSO, and the absorbance of the formazan solution was detected at 570 nm.
BrdU cell proliferation assays
TMEM97 knockout Hs578T and MDA-MB-231 cells and their parental wild-type counterparts were plated at 3×103 cells per well in 96-well plates, respectively. The BrdU incorporation assay was conducted using the Cell Proliferation ELISA BrdU Colorimetric Kit (Roche, Shanghai, China) according to the manufacturer’s instructions. Each group was performed in five replicates.
Colony formation assays
TMEM97 knockout Hs578T and MDA-MB-231 cells and their parental wild-type counterparts were seeded in a 6-well plate with a ratio of 500 cells per well. When the colonies were obviously visible, cells were washed twice with PBS, fixed with 4 % paraformaldehyde for 15 min, stained with crystal violet and photomicrography. To conduct quantitative analysis, colonies with a diameter larger than 30 µm were counted with ImageJ software. The experiments were replicated three times.
In vitro migration and invasion analyses
As described previously [24], TMEM97 knockout Hs578T and MDA-MB-231 cells and their parental wild-type counterparts were seeded in 24-transwell chambers with 8 µm pore membrane in serum-free medium with 1×105 cells. The lower chamber contained medium with 20% FBS. After incubation for 18–36 h, the cells were fixed with 4% paraformaldehyde for 15 min and then rinsed with PBS, followed by staining with 0.1% crystal violet. The unmigrated cells on the upper side of the membrane were rubbed away, and the migrated cells were photographed. Invasion assay was same as migration assay, except that the transwell chambers were precoated with Matrigel (Corning life science, Corning, NY, USA). To conduct quantitative analysis, the cells were eluted with 33% acetic acid and the absorbance was measured at 570 nm.
Sphere formation assays
TMEM97 knockout Hs578T and MDA-MB-231 cells and their parental wild-type counterparts were seeded at 500 cells per well in a 24-well plate with an ultra-low attachment surface containing DMEM/F12 medium (2% B-27, 10 ng/mL EGF, 10 ng/mL FGF, and 10 µg/mL insulin). After 10 days of culture, spheres with a diameter larger than 30 µm were counted, and representative areas were photographed using a light microscope. ImageJ software was used to analyze statistically the sphere numbers. The experiments were replicated three times.
Animal model study
All animal experiments were conducted by the protocols with the permission of Animal Research of Shenzhen University (permit number AEWC-201412003). For the breast cancer xenograft animal model, female BALB/c nude mice (6-week old) were purchased from Beijing Vital River Laboratory Animal Technology Company (Beijing, China). TMEM97 knockout MDA-MB-231 cells and the parental counterparts were implanted s.c. into the right flank of nude mice at a dosage of 1×107 cells per mouse. Following implantation, tumor growth was closely observed. Tumor volumes were measured with a caliper twice a week and calculated using the following formula: 0.528 × (length/2) × (width/2) [2]. CO2 euthanasia was performed when tumor size reached 10 mm in diameter. The tumors were excised, weighed and fixed in formalin for histological analysis. Mouse experiments were performed without knowledge of subject’s genotype.
Histological analyses
The tumors were fixed in formalin, embedded in paraffin, and sectioned. Hematoxylin and eosin (HE) staining and immunohistochemistry analysis were conducted as described previously [24]. The primary antibodies used are listed in Table S2.
Statistical analyses
Statistical analyses were performed using GraphPad prism 7.0 software (GraphPad, RRID: SCR_000306). Student’s t test was applied to compare the difference between two groups when the data showed a normal distribution. One-way analysis of variance (ANOVA) with Dunn’s multiple comparisons test was carried out to compare the means of several groups. The variance was similar between the groups that are being statistically compared. Data were presented as mean ± SD. Differences with P value <0.05 were considered statistically significant. All the experiments were replicated at least three times.
